Galactooligosaccharides (GOS) are a kind of oligosaccharides which cannot be digested and absorbed by the gastrointestinal tracts of human bodies, but directly enter the large intestine to be well utilized by various Bifidobacterium, and have special biological functions. GOS can improve the micro-ecological environment in the human body, aid multiplication of Bifidobacterium and other beneficial bacteria, and improve immunity of the human body. Meanwhile, GOS generate organic acids through metabolism to decline the pH value in tracts, to restrain the growth of salmonella and putrefying bacteria in the tracts, to reduce toxic fermented products and hazardous bacterial enzymes, to adjust gastrointestinal functions, thus reducing burdens on livers for decomposition of toxins. GOS have properties better than those of other functional oligosaccharides and therefore are more conveniently and easily applied to various fields as additives. GOS can be adapted to more food varieties and wider consumer groups, and have a huge application value and a huge market prospect.
GOS are usually prepared by five methods, namely extraction from natural materials, acid hydrolysis of natural polysaccharides, chemical synthesis, fermentation and enzymatic synthesis. GOS merely exist in nature, are color-less, have no charge, and therefore are difficultly extracted and separated. Products converted from the natural polysaccharides have a low yield, are complicated in elements and hard to be purified. The chemical synthesis tends to generate a lot of toxins and residues, causing serious environmental pollution. The fermentation method for producing the GOS is rarely studied, is still in the laboratory stage, and fails to realize mass production. At present, the industrial production of the GOS is completed through β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23). β-galactosidase, also called lactase, has dual activities, namely hydrolysis and transglycosylation. Previously, studies on the β-galactosidase mainly focus on utilization of the hydrolysis function thereof to produce low lactose milk products to relieve various side effects such as diarrhea and abdominal distension of lactose-intolerant patients caused by taking milk products. Since the special health-care functions of the GOS have been determined, production of the GOS by the transglycosidase effect of the β-galactosidase has become a study hotspot. The study mainly focuses on the three following aspects:
1. Screening of Strains for Generating the β-Galactosidase with High Transglycosidase Activity
Various microorganisms including yeasts, Bacillus, Aspergillus, Penicillium and Bifidobacteria, all have β-galactosidase with transglycosidase activity. Studies show that, due to different enzymatic properties, β-galactosidase coming from different sources vary with reactions conditions for synthesizing the GOS. β-Galactosidase can be classified into acidic type and neutral type according to the optimum pH values. Usually, β-galactosidase sourced from mold is acidic enzyme, with the best performance at a pH value in a range of 2.5-5.5 and at a relatively high temperature (50-60° C.); β-Galactosidase generated by yeasts and bacteria is neutral enzyme, with the best performance at a pH value in a range of 6-7.5 and at a relatively low temperature (30-40° C.). β-Galactosidase generated by different sources work on different substrates, and the types and ratios of oligosaccharide in the generated GOS are also diversified, so the new GOS verities emerge in endlessly. Even so, the screened β-galactosidase generally has low transglycosidase activity. Moreover, the highest yield of the GOS is usually 5-30%, failing to meet the demands of industrial production.
2. Optimization of Reaction Conditions and Improvement of Production Process
Some researchers tried to overcome the defect of low transglycosidase activity through optimizing the production conditions and processes of the GOS to enhance the yield of the GOS, and have made some achievements. Main methods include: increase in concentration of the initial lactose, control over the water activity using organic solvents and use of the immobilization technology. The hydrolysis and transglycosylation reaction of the β-galactosidase are inversible. When the substrate (lactose) concentration is low, the concentration of the hydrolysis product, namely galactose, is low, and the galactose has a limit effect on restraining the hydrolysis enzymatic activity. In such circumstances, the β-galactosidase represents high hydrolysis activity, while the transglycosylation activity is low, so the content of monosaccharides contained in the product is relatively high. When the lactose concentration is relatively high, the concentration of the hydrolysis product, namely the galactose, is relatively high, and the galactose reaching a certain value can restrain the hydrolysis enzymatic activity. The galactose is the substrate of the transglycosidase, and the high galactose concentration aids synthesis of galactooligosaccharide, and the product has a high content of oligosaccharide. Using organic solvents is good for composition of the oligosaccharide because organic solvents can reduce the water activity in the reaction system to affect the activity site and reaction mechanism of the enzyme, to induce the hydrolase to catalyze inverse transglycosylation, and to deviate the reaction balance from hydrolysis to oligosaccharide synthesis. Using the immobilization technology can greatly increase the pH and thermal stability of free enzymes, and can realize recycling and reduce production cost. Mozaffar was reported that β-galactosidase is absorbed to phenolic resin and then is cross-linked with glutaraldehyde, and then the yield of the oligosaccharide is enhanced by 20%. However, some studies find that when an immobilized enzyme is applied to the lactose solution with a relatively high concentration, the yield of the oligosaccharide is smaller than that the yield of the oligosaccharide generated when the free enzyme is used. Thus it can be seen that problems cannot always be radically solved simply by optimizing conditions.
3. Genetic Engineering to Enhance Expression of the β-Galactosidase and to Improve its Properties
In the natural world, the yield of GOS by wild β-galactosidase is generally maintained in the range of 20-45%. The low yield fails to meet production demands, screening excellent transglycosidase mutant enzyme through molecular modification has become a research hotspot. Hansen O. (2001) found that after Bifidobacteria β-galactosidase BIF3 is deleted with 580 amino acids at a C-terminal, the protein is converted into an efficient transglycosidase which can generate GOS by using almost 90% of lactose, while hydrolysis elements account for 10%. When the lactose concentration is in the range of 10%-40%, the ratio of the transglycosidase activity to the hydrolysis activity is always maintained at 9:1. In 2009, Placier G. carried out directed revolution on the β-galactosidase sourced from Geobacillus stearothermophillus KVE39, and successfully obtained three strains of mutants R109W, R109V and R109K on the screen strategy of enhancing the transglycosidase activity while reducing the hydrolysis activity. In 18% (w/v) lactose, the yield of oligosaccharide generated by three mutants was 23%, 11.5% and 21%, respectively. In wild enzyme, the oligosaccharide yield was 2% only. Wu Y. (2013) modified molecules of the β-galactosidase sourced from Sulfolobus acidocaldarius to study the most appropriate generation conditions of GOS. Under respective most appropriate conditions, the GOS yield of the mutant F441Y was 61.7%, F359Q was 58.3%, and the wild enzyme was 50.9%.
However, so far, the screening and separation as well as process optimization of the natural enzymes, and genetic engineering to enhance the expression of the β-galactosidase and improve properties both fail to change the current states of low transglycosidase activity and low yield of the β-galactosidase, resulting in low synthesis yield of the GOS and extremely high production cost which seriously restrain the low-cost production, promotion and application of the GOS.
Therefore, creating a novel β-galactosidase with high transglycosidase activity and low-cost production are two of main problems to be solved urgently in the current research and production.